Immunoproteomics: The Key to Discovery of New Vaccine Antigens Against Bacterial Respiratory Infections.

2012·0.55 MB·English

by  Dennehy, Ruth

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Current Protein and Peptide Science, 2012, 13, 807-815 807
Immunoproteomics: The Key to Discovery of New Vaccine Antigens
Against Bacterial Respiratory Infections
Ruth Dennehy and Siobhán McClean*
Centre of Microbial Host Interactions, Centre of Applied Science for Health, Institute of Technology Tallaght, Old
Blessington Road, Dublin 24, Ireland
Abstract: The increase in antibiotic resistance and the shortage of new antimicrobials to prevent difficult bacterial infec-
tions underlines the importance of prophylactic therapies to prevent infection by bacterial pathogens. Vaccination has re-
duced the incidence of many serious diseases, including respiratory bacterial infections. However, there are many patho-
gens for which no vaccine is available and some vaccines are not effective among all age groups or among immunocom-
promised individuals. Immunoproteomics is a powerful technique which has been used to identify potential vaccine can-
didates to protect against pathogenic bacteria. The combination of proteomics with the detection of immunoreactive anti-
gens using serum highlights immunogenic proteins that are expressed during infection. This is particularly useful when
patient serum is used as the antigens that promote a humoral response during human infection are identified. This review
outlines examples of vaccine candidates that have been identified using immunoproteomics and have successfully pro-
tected animals against challenge when tested in immunisation studies. Many immunoreactive proteins are common to sev-
eral unrelated pathogens, however some of these are not always protective in animal immunisation and challenge studies.
Furthermore, examples of well-established immunogens, including Bordetella pertussis antigen FHA were not detected in
immunoproteomics studies, indicating that this technology may underrepresent the immunoreactive proteins in a patho-
gen. Although only one step in the pathway towards an efficacious approved vaccine, immunoproteomics is an important
technology in the identification of novel vaccine antigens.
Keywords: Antigens, immunoproteomics, immunoreactive proteins, respiratory disease, vaccine, virulence.
INTRODUCTION species has limited the potential of LPS as potential vaccine
antigens, for example P. aeruginosa LPS [2]. Many ap-
The value of vaccination as a means of combating life-
proved vaccines are glycoconjugate in nature (e.g. vaccines
threatening and chronic infectious diseases is well estab-
for Neisseria meningitidis and Streptococcus pneumoniae)
lished. Many diseases which previously contributed to mor-
and have proved effective against certain serogroups of
tality are now prevented by vaccination. However, according
pathogens; but they tend to suffer from the same issues with
to the WHO, about 1.5 million of deaths among children
variability across strains. In addition, serotype switching and
under five in 2008 were caused by vaccine preventable dis-
serotype replacement may undermine their effectiveness in
eases. In addition many approved vaccines are more effec-
the future [3]. Proteins are often more conserved across bac-
tive in young children than in adolescents and adults. Given
terial strains and have more potential to protect against sev-
that infectious diseases are still the leading global cause of
eral strains of the same bacterial species.
morbidity and mortality and when considered in the context
of both the rise in antimicrobial resistance and the lack of Investigation and harnessing of the host-pathogen re-
novel effective antibiotics in the drug development pipeline sponse are beneficial to two separate but essential aspects to
[1], the need for more efficacious vaccines for bacterial vaccine development. Exploitation of the human humoral
pathogens cannot be underestimated. response by determining immunogenic bacterial protein us-
ing sera from vaccinated, colonised or recovering individuals
There are several bacterial cell components incorporated
has played an important part in identifying novel vaccine
into subunit vaccines, LPS, glycoconjugated vaccines, DNA
antigens. Identification of novel antigens is only part of the
vaccines, protein vaccines, which in addition to live attenu-
process of vaccine development, therefore examination of
ated vaccines and whole killed cell vaccines stimulate the
the host response in adults and in human volunteers at the
host immune response in a variety of ways. Vaccine studies
cellular and humoral response level is also essential in the
have often focussed on lipopolysaccharide (LPS) due to its
development of more efficacious vaccines. Protection against
potent stimulation of the immune response; however the an-
certain intracellular pathogens requires a strong cell-
tigenic diversity of the O-antigen component of LPS within
mediated immune response driven by polarisation towards a

Th1 response; while other extracellular pathogens are best
*Address correspondence to this author at the Centre of Microbial Host
protected by a Th2 response which promotes antibody pro-
Interactions, Centre of Applied Science for Health, Institute of Technology
Tallaght, Old Blessington Road, Dublin 24, Ireland; Tel: [phone]; duction and antibody-mediated cell killing [4]. Indeed
Email: [email protected] knowledge of the interactions between pathogens and host
1875-5550/12 $58.00+.00 © 2012 Bentham Science Publishers
808 Current Protein and Peptide Science, 2012, Vol. 13, No. 8 Dennehy and McClean
cells in vitro is also critical, where the bacterial components is not optimal for protection in children under two or in im-
that attach to host cells are investigated and elucidated. This munocompromised patients [3]. Furthermore, there has been
review will focus on a number of examples of the exploita- serotype replacement and increased incidence of non-vaccine
tion of the host response in the identification of novel anti- serotypes, including the antimicrobially resistant serotype
gens and in the development of identified vaccine antigens as 19A, which now predominates in some countries [3]. Al-
prophylactic therapies against respiratory bacterial diseases. though the recently approved 10- and 13-valent vaccines
target more serotypes and prevent invasive disease in chil-
EXPLOITING THE MICROBIAL–HOST RESPONSE dren, there are at least 92 serotypes. This means that identifi-
TO IDENTIFY NEW ANTIGENS cation of protein antigens which are more highly conserved
across the species than the capsular polysaccharides may
Reno Rappuoli and co-workers pioneered the use of a
present more effective alternatives.
genomics approach to identify novel antigens against Neisse-
ria meningitidis serogroup B using reverse vaccinology, in Immunoproteomics has been used to identify potential
the late 1990s. They sequenced the genome of the pathogen, immunogenic antigens in S. pneumoniae. Adults develop
identified proteins which were likely to be surface associ- antibodies to S. pneumoniae during asymptomatic carriage
ated, expressed these in Escherichia coli and administered and it has been shown that the antibody response is age de-
the 350 proteins to mice to identify those that were immuno- pendent [7,8]. This increase in antibody response to S.
genic [5]. The sequencing step of the process took 18 months pneumoniae proteins which correlated with a reduction in
at the time. The development of genomics since then has morbidity in children prompted Ling et al., to focus on anti-
meant a rapid expansion in the capabilities to identify new gens which showed increased immunogenicity with age of
antigens. Over 2,800 bacterial genomes have been com- the child [9]. Two-dimensional Western blotting of cell wall
pletely sequenced at time of writing, a figure which is in- fractions from S. pneumoniae probed with sera from healthy
children ranging in age from 18 to 42 months or with sera
creasing week on week (NCBI Microbial genomes:
from adults highlighted seventeen immunoreactive proteins.
www.ncbi.nlm.nih.gov/genome). This vast bank of informa-
Two of these, both metabolic enzymes, were equally protec-
tion combined with the advances in proteomics, including,
tive against two genetically different strains in a mouse chal-
the relatively reproducibility and ease of preparing 2-D gels
lenge model. While this indicates promise for the proteomic
has meant a rapid increase in proteomic analysis of bacterial
approach to identify protective antigens, the protection ob-
pathogens which has given insights into mechanisms of bac-
tained was only 36%. Another two antigens identified in this
terial virulence and pathogenesis, adaptation during chronic
study, 6-phosphogluconate dehydrogenase (6-PGD) and Glu-
colonisation and host response. The natural step up to im-
tamyl-tRNA synthase were subsequently shown to be in-
munoproteomics, whereby 2-D blots are probed with host
volved in adhesion to A549 cells [10,11]. While both inde-
serum following infection or immunisation has greatly en-
pendently protected mice against a lethal challenge, the level
hanced the identification of potential vaccine candidates, by
of protection was only marginally better than that observed
enabling the discovery of novel proteins that stimulate the
with GAPDH and Fructose-bis-phosphate aldolase. In the
humoral host immune system. A key advantage of this ap-
case of 6-PGD, immunization also reduced lung and blood
proach is that final expressed bacterial proteins are used in
colonisation levels by several fold. The latter antigen, glu-
the analysis, which have been completely processed and
tamyl-tRNA synthase shows promise due its lack of a human
post-translationally modified by the pathogen. Several
orthologue, the former protein has high homology to the hu-
groups have used this method to discover potential vaccine
man orthologue which could be detrimental to its develop-
candidates with varying degrees of success. Different sera
ment as a protective antigen [11].
have been used in these types of studies which range from
using colonised patient sera, convalescent sera, or sera from A separate proteomic analysis of cell wall proteins identi-
challenged mice or rabbits. Furthermore, the majority of fied proteins which were expressed across at least 40 S.
immunoproteomic studies have focussed on cell surface pro- pneumoniae serotypes and during animal infection [12]. Five
teins or outer membrane proteins as this subgroup of bacte- proteins were evaluated in a mouse sepsis model, and al-
rial proteins represent the interface for host pathogen interac- though lung bacterial burdens were reduced following indi-
tions and regularly include adhesins. Other groups have ex- vidual immunisations with two of the proteins, these did not
amined secreted proteins as they are also primary antigen prolong the survival of mice. The poorer outcome in this
targets of the host immune response and include many viru- study, compared with that of Ling and co-workers, may be
lence factors. Collectively, their exposure to the host im- due to the fact that immunogenic proteins were not necessar-
mune system marks these pools of proteins as a rich source ily selected for this study. More recently, immunoproteomic
of vaccine candidates. analysis of the S. pneumoniae secretome also identified anti-
gens which were shown to be immunogenic using immu-
nised rabbit serum [13]. In total 54 proteins were detected, of
Streptococcus Pneumoniae
which only 23 were identified, including several proteins
Streptococcus pneumoniae is a Gram positive bacterium which were known virulence factors, in addition to novel
that is a leading cause of bacterial pneumonia, meningitis proteins. These have the potential as diagnostic markers in
and pneumococcal septicaemia. It accounts for 20 to 50% of addition to vaccine candidates. Many of these immunogenic
community acquired pneumonia cases and is the cause of proteins had been previously identified in other species and
over 820,000 deaths each year in children under five [6]. The in some cases by Ling et al., e.g., Enolase, ZmpB, serine
seven-valent glycoconjugate pneumococcal vaccine Prevnar protease [9]. However, vaccine studies on these have not
7 has reduced the incidence of invasive pneumococcal dis- been reported to date.
ease in children and mortality from this disease; however, it
Immunoproteomics: The Key to Discovery of New Vaccine Antigens Current Protein and Peptide Science, 2012, Vol. 13, No. 8 809
Overall, the comparable survival rates following lethal fying immunoreactive antigens in B. pseudomallei [20]. This
challenge (36 to 40%) for the four separate antigens de- functional genomic approach allowed the screening of 5760
scribed above and the lack of improved survival for others open reading frames which were then used to genetically
may suggest that more knowledge of the specific protective immunise mice. Three clones were selected from an initial
host response to S. pneumoniae is required to optimise fur- screen of 14 serum reactive phagemids, based on their strong
ther vaccine development. Indeed the finding that four dis- reactivity. Serological evaluation of 74 patients showed that
tinct antigens showed very comparable survival rates may be 94% of patients were seropositive for one of these proteins,
a feature of the animal model used. It is possible that mice OmpA. B. pseudomallei have several putative OmpA genes,
are not predictive of the immunological response to S. pneu- cloning of these resulted in 6 recombinant proteins. Two of
moniae in humans. This is a key limitation of pre-clinical these were followed up, Omp3 which clustered with the
vaccination studies that must be always considered. An ex- OmpA of E. coli and Klebsiella pneumonia OmpA following
ample of this is highlighted below in the discussion on Bor- multiple sequence alignment, and Omp7 which was closely
detella pertussis immunoproteomics studies. related to OmpA of Porphyromonas gingivalis [20]. Subse-
quent mouse immunisation with these resulted in 50% pro-
Burkholderia Pseudomallei and other Members of Genus tection against a lethal challenge for two different recombi-
Burkholderia nant OmpA proteins with the most reactivity with sera from
melioidosis patients [21]. When this pilot immunisation
Burkholderia pseudomallei, the causative agent of
study is considered together with the widespread seropositiv-
melioidosis, is a Gram-negative, intracellular pathogen en-
ity for OmpA among convalescent melioidosis patients, it
demic in Southeast Asia and Northern Australia. The disease
strongly suggest the potential for this antigen as one compo-
is considerably variable in humans ranging from acute septi-
nent of a future multi-valent vaccine against melioidosis.
caemia, pneumonia to chronic or latent infection which can
Using a similar approach, Su et al., carried out a genomic
persist and emerge decades later. The bacterium is intrinsi-
survey of B. pseudomallei, screening an expression library
cally antibiotic resistant and has a mortality rate up to 50%;
with sera from melioidosis patients [22]. Among the 109
however despite its significant morbidity and mortality, there
proteins identified from seropositive clones, one protein was
is currently no licensed vaccine against this infection. Hard-
selected, OMP85, for later study, as it is a highly conserved
ing et al., have used serum from convalescent patients to
protein. Intraperitoneal immunisation of mice with this re-
probe 2-D blots of B. pseudomallei OMPs [14]. By biotiny-
combinant antigen resulted in 70% protection of mice over
lation of the bacteria prior to OMP preparation, they were
15 days, as opposed to 10% in non-immunised mice in addi-
able to focus solely on surface expressed proteins. They
tion to significantly reduced bacterial load in lungs and
identified nine surface proteins which were immunoreactive,
spleen [23].
including elongation factor Tu (EF-Tu), polyribonucleotide
nucleotidyl transferase and two chaperones, GroEL and Most recently, protein microarrays have been used to
DnaK. Among the biotinylated proteins identified were sev- identify immunoreactive proteins with plasma from recov-
eral that would be considered to be cytoplasmic. This high- ered melioidosis patients or seropositive healthy individuals
lights a common theme in proteomic analysis, that proteins [24]. Of the 27 antigens identified, seven were more strongly
predicted to be cytoplasmic and without any signal peptide recognised by plasma from recovered individuals over
are exposed at the bacterial surface [15]. A later study util- healthy controls. These represented antigens which were
ised rabbit serum to probe 2-D blots of membrane proteins previously identified, and/or associated with virulence, in-
and again identified elongation factor Tu (EF-Tu) and DnaK cluding flagellin and flagellar hook associated protein,
among the immunoreactive proteins of a less virulent related OmpA and BipB, confirming the capacity for patient serum
species, B. thailandensis, which shares 94% identity at the to identify potential vaccine candidates. Although BipB was
amino acid level with B. pseudomallei [16]. EF-Tu was previously shown not to prolong survival of challenged mice
shown to be secreted in outer membrane vesicles (OMVs) [25], antibody levels against OmpA (one of the two OmpAs
which are shed from B. pseudomallei during in vitro growth. which was also found to be protective by Hara et al. [21])
Mucosal immunisation of mice with EF-Tu resulted in the were 10-fold higher in patients that had only one episode of
generation of antigen specific IgG and IgA, polarised Th1 melioidosis relative to those with recurrent disease, again
immune response and significantly reduced the bacterial indicating that antibodies specific to this protein may be pro-
burden in the lungs following a lethal challenge with B. thai- tective. Another closely related pathogen, Burkholderia ce-
landensis. OMVs are gaining momentum as a potential vac- pacia is one of 17 closely related species that comprise the
cine platform [17,18]. These are engineered proteoliposomes Burkholderia cepacia complex [26]. This group of pathogens
which incorporate the vaccine antigen, adjuvant in a particu- are responsible for chronic lung infections in people with
late carrier. In a later study sub-cutaneous immunisation of cystic fibrosis and other immunocompromised individuals.
mice with purified B. pseudomallei OMVs resulted in protec- The secretory proteins of B. cepacia were analysed on 2-D
tion of 60% of mice against a lethal challenge with high blots probed with serum from immunised mice [27]. One
OMV-specific titres, together with T-cell responses and hu- protein in particular showed a 96% homology with a metal-
moral responses which were suggestive of a Th1 cell- loprotease from a B. pseudomallei strain and it was sug-
mediated immunity [19]. It was not stated whether these pu- gested that this could cross-protect against both pathogens.
rified OMVs contained EF-Tu or what their antigenic com- No in vivo challenge experiments were reported.
position was.
Overall, investigation of immunoreactive proteins in B.
Immunoscreening of a lambda genomic library with pseudomallei using immunoproteomics or gene expression
pooled patient serum represents an alternative way of identi- libraries has already allowed the identification of vaccine
810 Current Protein and Peptide Science, 2012, Vol. 13, No. 8 Dennehy and McClean
candidates to protect against melioidosis, and further identi- However, while vaccination decreased the incidence of this
fication of additional antigens should allow the development infection, there are reports of a resurgence of pertussis
of a multivalent vaccine which will improve protection among adults and adolescents in recent years, in developed
against B. pseudomallei and eliminate bacterial persistence. countries, despite good uptake of pertussis vaccination [30].
Taking both chronic infection and the intracellular lifestyle The current 5-component acellular vaccine administered in
of B. pseudomallei into consideration, it is evident that there combination with diphtheria and tetanus toxoids (DTaP) and
will be significant challenges in the development of an effec- also combined with Haemophilus influenzae B and polio
tive vaccine against this agent. A successful vaccine is likely antigens, contains five Bd. pertussis antigens. The antigens
to need the induction of both humoral and cell-mediated im- include pertussis toxoid, filamentous hemagglutinin (FHA),
munity for the complete protection against this pathogen. fimbriae 2 and 3 and pertactin. These antigens are all viru-
lence factors and play a role in bacterial attachment (re-
Neisseria meningitidis viewed by Marzouqi et al. [31], and antibodies to these have
been identified in infected patients [32]. There is renewed
N. meningitidis colonises the upper respiratory tract,
interest in improving pertussis vaccination which has led to a
causing a mild respiratory infection, however in about 15%
number of immunoproteomics studies. These, however,
of patients it invades. There are five serogroups of this Gram
highlight some limitations of immunoproteomics. Two sepa-
negative pathogen which causes pneumonia, meningococcal
rate studies of Bd. pertussis surface proteins [33] and secre-
meningitis and septicemia. Glycoconjugate vaccines are
tome [34] were carried out using mouse antisera from inacti-
available which are effective against only four serogroups
vated Bd. pertussis immunised mice as the primary antibody.
and it is widely recognised that the disease can be prevented
The surface immunoproteomes for two strains of Bd. pertus-
by vaccination to induce antibody dependent complement
sis were nearly identical. Among the 27 immunoreactive
mediated bactericidal activity [28]. No vaccine exists for
proteins identified across both strains, pertactin which is a
serogroup B, which is responsible for over 30% of meningo-
component of the current approved pertussis vaccine, was
coccal disease in the US and up to 80% in Europe. In what
identified. Interestingly, although FHA is present in the sur-
was the first application of reverse vaccinology, several se-
face proteome and is a highly immunogenic component of
rogroup B antigens were identified which resulted in high
acellular pertussis vaccines, it was not identified as an im-
titres of bactericidal antibodies, including, factor H binding
munogenic protein among both lab strains examined in this
protein (fHbp) and Neisserial heparin binding antigen
study [33]. This may be due to its very large molecular size
(NHBA) [5]. Three highly immunogenic antigens (fHbp,
and consequent poor transfer to blotting membranes. An-
Neisseria adhesin (NadA) and NHBA were combined with
other study of immunoreactive proteins in Bd. pertussis ana-
OMVs from an epidemic strain and are currently in phase III
lysed the serum of children that had been immunised with
clinical trials. In contrast to the studies on other pathogens, a
the whole cell pertussis vaccine also identified pertactin
recent immunoproteomic analysis of Neisseria meningitidis
among the immunoreactive proteins, but neither FHA nor
failed to identify potentially protective antigens, i.e antigens
pertussis toxin. Zhu et al., compared the murine immunopro-
which produced bactericidal antibodies when subsequently
teome with the human immunoproteome of Bd. pertussis and
tested in mice [29]. Sera from both acute and convalescent
found considerable differences in immunoreactivity [35].
patients were used to probe 2-D blots allowing the identifica-
Only four human immunoreactive proteins were detected
tion of 33 immunoreactive proteins. Interestingly many of
from immunised or infected mice and many human immu-
the proteins identified were also shown to be immunogenic
nodominant antigens were not recognised by murine immune
in other respiratory pathogens, e.g. GroEL, elongation factor-
sera. This clearly demonstrates that bacterial antigens may be
Tu, DnaK and cysteine synthase, but comparison of different
recognised differently by the murine and human immune
patient sera indicated that meningococcal patients have very
systems and may indicate that researchers should be cautious
variable immune responses against a range of antigens.
when using murine antisera, or indeed antisera from other
When the recombinant proteins were expressed and adminis-
species, in the identification of potential human vaccine anti-
tered to mice, all sera had significant protein-specific ELISA
gens.
titres, however none of these sera showed bactericidal activ-
ity. Protection of mice against challenge was not reported.
INVESTIGATING THE HOST RESPONSE TO EN-
HANCE THE EFFICACY OF IDENTIFIED ANTI-
B. Pertussis
GENS: DEVELOPMENT OF A P. AERUGINOSA
Bordetella pertussis is a Gram-negative bacterium re- VACCINE
sponsible for the highly contagious respiratory infection, The identification of vaccine antigens by immunopro-
whooping cough (pertussis), in humans. Pertussis vaccina- teomics or other immunomic analysis, although powerful
tion exemplifies the benefits of vaccination and the issues technologies, is only the first step in a long process to suc-
that arise when vaccine uptake wanes. The whole cell pertus- cessful protective vaccine. Indeed, several immunogenic
sis vaccine had a major impact on reducing childhood mor- proteins identified have failed to protect against challenge in
tality when it was introduced in the 1940s, however, serious subsequent animal models (for example, BipB in B. pseu-
side effects, including encephalitis, in a subgroup of vacci- domallei [25] and GroEL and EF-Tu in N. meningitidis [29].
nated children led to its reduce uptake and consequent wide- A full knowledge of the host response that is required for a
spread outbreaks throughout the 1970. Introduction of the protective response in humans is essential in the develop-
acellular pertussis vaccine improved confidence and conse- ment of an effective vaccine. The process involved is exem-
quently uptake of the pertussis vaccination with the effect of plified by the separate development of vaccine candidates
reducing the incidence among children for the next decade. against P. aeruginosa. This is an opportunistic pathogen
Immunoproteomics: The Key to Discovery of New Vaccine Antigens Current Protein and Peptide Science, 2012, Vol. 13, No. 8 811
which colonises 80% of adults with the genetically inherited response studies have been carried out on the proteins in-
disease cystic fibrosis, contributing to the mortality and mor- volved in this OprF-OprI hybrid antigen. OprF has been
bidity of that disease. It is also responsible for a significant shown to involved in attachment of P. aeruginosa to host
proportion of ventilator associated pneumonias and colonises cells [46] and is upregulated during chronic infection, in con-
and invades burn patients causing sepsis. P. aeruginosa trast to other virulence factors which are either lost or down
strains are characterised by intrinsic resistance to a range of regulated [47]. OprF is involved a range of other virulence
antimicrobial agents and antibiotics and have multiple characteristics in P. aeruginosa, including Type III secretion
mechanisms of resistance which means that treatment is dif- system, production of virulence factors such as pyocyanin,
ficult. In susceptible populations, prevention of infection elastase and exotoxin A and adhesins [46], again illustrating
would represent a better approach to therapy and indeed, the potential of virulence factors and adhesins as protective
prevention of infection with P. aeruginosa is a major objec- antigens. It has been suggested that OprF is a host immune
tive in the treatment of chronic obstructive pulmonary dis- system sensor enabling P. aeruginosa to enhance virulence
ease and cystic fibrosis. The development of protective vac- when bacteria are in contact with the host [46].
cines to protect susceptible populations against this pathogen
Independently, another aspect of the P. aeruginosa at-
spans several decades. The majority of the work has fo-
tachment has been exploited, leading to the development of
cussed on a limited number of antigens that showed promise
flagellin and flagella-based vaccines which have been tested
early in their development. The potential for outer membrane
in Phase III clinical trials. Most CF patients immunised de-
protein OprF was first identified as a protective antigen as
veloped high serum titres to flagella A and B and a signifi-
early as 1984 [36] and its suitability confirmed when it was
cant reduction in the number of immunised patients that de-
demonstrated using a panel of monoclonal antibodies as be-
veloped P. aeruginosa infection was observed among the
ing a conserved protein, with several conserved surface epi-
group of patients that received all four vaccinations [48].
topes [37]. It was evaluated as a hybrid protein with another
Disappointingly, the study did not show a significant reduc-
conserved outer membrane protein OprI, which was also
tion in chronic infection in these patients, or an alteration in
antigenically cross-reactive against seventeen serogroups
the rate of decline in lung function. In a follow-on analysis
[38]. This recombinant hybrid protein was subsequently
of the immune response to vaccination, Campodonico et al.,
shown to protect immunosuppressed mice against P. aerugi-
showed that the polymeric flagella rather than monomeric
nosa challenge and was shown to be tolerated in human vol-
flagellin appeared to be superior for generating immunity to
unteers [39]. Antibodies against OprF have been shown to be
P. aeruginosa. Antibodies to type a and b flagella were more
protective in burn and ocular models of P. aeruginosa infec-
potent in mediating opsonic killing of P. aeruginosa and
tion [40,41], however, only partial responses have been
mediating passive immunity in mice than serum raised to
achieved in cystic fibrosis patients, so the recent focus of
flagellin. This was attributed to flagellin inducing high titres
development of this vaccine antigen has been in understand-
of antibodies which could neutralise the innate immunity due
ing the host response and optimising the adjuvant/ vaccine
to TLR5 activation [49].
administered to maximise the protective response. Investiga-
Combinations of both outer membrane proteins OprF and
tion of the host response in chronically colonised CF patients
has suggested that a Th2 response correlated with infection, OprI and flagellins have also been evaluated in a multimeric
while a Th1 cell response correlated with an improved out- fusion protein vaccine, in order to further optimise the pro-
come and may be more protective [42,43]. Bumann et al., tective host response by exploiting the ability of flagellin to
activate TLR-5 and act as an adjuvant as well as an antigen
have shown that a live vaccine constructed from a number of
[50]. Immunisation of mice with a combination of an OprF
attenuated Salmonella vaccine strains induced mucosal re-
epitope fused with OprI incorporated at the N-terminus of
sponses in human volunteers when administered orally or
nasally [44]. This phase I/II clinical trial on the OprF-OprI the type A or type B flagellin resulted in high level of anti-
conjugate vaccine demonstrated high levels of specific anti- bodies to OprF, flagellin and OprI. The Opr/flagellin recom-
bodies, including IgA at the bronchial surface and showed binant proteins elicited high titres of IgG2a, in addition to
IgG1, although the response was biased towards a Th1 re-
that the nasal route was superior in terms of mucosal anti-
sponse. In addition, immunisation reduced the bacterial bur-
body response [44]. While this work towards an OprF-OprI
den in mice challenged with non-mucoid P. aeruginosa
vaccine to protect children with CF against P. aeruginosa is
promising, a Phase III randomised control trial will be neces- strains. One limitation of this study was that although anti-
sary to determine its efficacy and to demonstrate that anti- gen specific antibodies were able to activate complement,
body titres in this case, correspond to protection. An alterna- complement-mediated killing was only effective against non-
mucoid bacteria, being less effective against mucoid bacte-
tive immunisation approach using adoptive transfer of OprF-
ria. The conversion of P. aeruginosa to a mucoid phenotype
pulsed dendritic cells as the vaccine in a mouse model
during chronic infection which may have significant impacts
showed significant protection and clearance of P. aeruginosa
and activation of a Th1 response with a concomitant reduc- on the exposure and expression of surface antigens and is
tion in inflammatory cell recruitment. They also observed likely to be an additional challenge in developing effective
protection against a mucoid strain which is generally associ- vaccines to protect against this pathogen.
ated with chronic colonisation of the CF lung, albeit a There are considerable advances yet to be made both in
slightly delayed response relative to the non-mucoid strain our knowledge of the host response and in the development
[45]. of an effective vaccine for this difficult pathogen, again
Understanding the host pathogen interaction of these demonstrating that antigen identification is only a fraction of
vaccines will allow the development of future vaccines the process to a successful vaccine. These studies also high-
which protect against different pathogens. Extensive host light the limitations of immunoproteomics to predict good
812 Current Protein and Peptide Science, 2012, Vol. 13, No. 8 Dennehy and McClean
Table 1. Examples of Immunoproteomics Studies to Identify Vaccine Antigens and/or Virulence Factors in Respiratory Pathogenic
Bacteria
Pathogen # Antigen(s) of interest Serum source Protein fraction Protective Comment Ref
Immunised chil- Total membrane Observed differ-
Pertactin, serum resistance protein
dren (whole cell proteins & ex- ences between
Bd. pertussis 30 (BrkA), OmpP, sulphate binding protein NA [35]
pertussis vac- tracellular pro- mouse and hu-
(Sbp)
cine) teins man serum data
immunodomi-
Bd. pertussis 1 Peptidoglycan associated lipoprotein Immunised mice Cell lysate NA [62]
nant antigen
Serum resistance protein, pertactin, HSP-
Comparison of
60, HSP70,Clp protease, serine protease,
Bd. pertussis 25 Immunised mice Soluble proteins NA two vaccine [34]
EF-Tu, Glutamyl-tRNA aminotrans-
strains
ferase, phosphoenolpyruvate synthase
Serum resistance protein, pertactin, sero- Comparison of
Bd. pertussis 11 type fimbral subunit, HSP-60, HSP-10, Immunised mice Surface proteins NA two vaccine [33]
ATP synthase strains
EF-Tu, pyruvate carboxylase, ATP-
dependent zinc metalloprotease, tyrosyl-
B. cepacia 18 Immunised mice Secreted proteins NA [27]
tRNA synthase, phospholipid glycerol
acyltransferase, ABC efflux pump
9 Antigens both
GroEL, DnaK, PhaP, Ef-Tu, polyribonu- Immunised rab-
B. pseudomallei 12 Surface proteins NA biotinylated and [14]
cleotide nucleotidyltransferase bit serum
immunoreactive
Rabbit from B. EF-Tu
Ef-Tu, DnaK, AhpC, oxodipate CoA
B. thailandensis 16 mallei immu- Protection immunoprotec- [63]
succinyltransferase, HSP10,
nised rabbits tive
F1F0 ATP synthase, EF-Tu, glutamylt-
Tularemia pa-
RNA synthase, fructose bisphosphateal-
F. tularensis 31 tients and vacci- NA [64]
dolase, GAPDH, pyruvate dehydro-
nated individuals
genase, DnaK
Comparison of
responses from
successful vac-
EF-Tu, GroEL, ATP synthase, DNA Mouse sera from Soluble and cination and
F. tularensis 9 gyrase, chitinase family 18 protein, DNA Live attenuated membrane en- NA unsuccessful [65]
directed RNA polymerase immunised mice riched vaccination –
examples of
successful vac-
cination in bold.
Membrane pro-
teins, in vitro
Leptospira
Lipoprotein L21, L41, L32. Loa22. Rat infection culture and NA [66]
interrogans
guinea pig infec-
tion
PasP protease, zinc dependent aminopep- Compared 2
P. aeruginosa 51 tidase, azurin, OprH, LasB protease, CF patient sera Extracellular NA strains, PA01 [51]
PrpL protease and PA14
No serum
TufA (EF-Tu), alcohol dehydrogenase, bacterici-
Convalescent
N. meningitidis 33 DnaK, cysteine synthase, aconitasehy- Total protein dal anti- [29]
patients
dratase, ATP synthase F1, Opa900 bodies
produced
Immunoproteomics: The Key to Discovery of New Vaccine Antigens Current Protein and Peptide Science, 2012, Vol. 13, No. 8 813
(Table 1) Contd….
Pathogen # Antigen(s) of interest Serum source Protein fraction Protective Comment Ref
Glutamyl- Healthy chil-
4 tested, all
tRNAamidotransferase,GAPDH, dren and adults
S. pneumoniae 17 Surface proteins showed [9-11]
fructose bis-phosphate aldolase, 6- (asymptomatic
protection
PGD carriage)
Enolase, serine protease, N-
acetylglucosamine-6-phosphate All 6 listed immunore-
deacetylase, general stress protein, Immunised No in vivo active proteins found in
S. pneumoniae 23 Secreted proteins [13]
pneumococcal histidine triad pro- rabbits studies all 3 clinical strains
tein D precursor , Zinc metallopro- tested.
tease
# Number of antigens identified.
vaccine antigens. Although flagellin type B protein (FliC) the studies outlined here are metabolic enzymes and would
was identified in an immunoproteomics analysis of extracel- be considered to have a cytoplasmic localisation within the
lular P. aeruginosa proteins using CF patient serum [51], cell. The fact that they are immunogenic during infection in
confirming its potential to produce high antibody titres; nei- animals and patients, could be due to bacterial cell lysis or
ther OprF nor OprI were identified in this study. The authors could be due to these enzymes having as yet an undeter-
attributed the presence of flagellin and other OMPs among mined role in bacterial virulence and are worthy of further
extracellular immunogens to bacterial cell lysis. However, study.
OprF and OprI would also be expected to be present in a In summary, the advances in immunoproteomics have led
bacterial lysate. Their lack of detection in this study could to a massive increase in the potential vaccine candidates
suggest that they were not released from the cell surface, which may be useful to protect against difficult to treat bac-
under the conditions tested, or like certain Bd. pertussis anti- terial pathogens. While this review has focussed on respira-
gens these may not be amenable to detection by immunopro- tory pathogens, many immunoreactive antigens have also
teomics. been identified using this approach for other bacterial patho-
gens, e.g. Staphylococcus epidermidis [60], Campylobacter
CONCLUSIONS concisus [61]. As outlined, this potent technology is not
without limitations, but when used with serum from either
Examples of immunoreactive proteins identified by im-
colonised or convalescent patients, it is particularly useful as
munoproteomics in a range of respiratory pathogens are
it allows researchers to identify immunoreactive antigens
listed in (Table 1). Many of these are common across several
that are expressed during human infection. Further thorough
unrelated bacteria. For example, GroEL (HSP60) and DnaK
examination of the host immune response, including immu-
(HSP70), are both conserved heat shock proteins and are
nisation studies in a number of carefully selected models,
highly immunoreactive in several diverse pathogens includ-
will help identify which of the immunogenic proteins can be
ing Shigella flexineri, Helicobacter pylori and Yersinia en-
tercolitica [52-54]. Immunisation with GroEL, but not DnaK taken further to human trials. It is likely that multivalent vac-
resulted in 100% protection in mice following Bacillus an- cines can be developed by careful selection of immunoreac-
thracis challenge [55]. GroEL immunisation was also protec- tive antigens identified with immunomic technologies and
tive against S. pneumoniae [56]. EF-Tu is another antigen when combined with the appropriate adjuvant will increase
which is immunoreactive across several pathogens. Interest- our weaponry in the defence against respiratory bacterial
ingly, while it was protective against B. thailandensis, it was infections.
not protective when used in immunisation studies followed
by challenge with N. meningitidis. In addition, it was among CONFLICT OF INTEREST
the immunoreactive proteins that were identified with serum
The author(s) confirm that this article content has no con-
from both successfully and unsuccessfully vaccinated mice
flicts of interest.
using an attenuated Francisella tularensis vaccine strain,
suggesting that antibodies against EF-Tu are not protective
ACKNOWLEDGEMENTS
against this pathogen [65]. EF-Tu has been shown to bind to
host proteins such as fibronectin, mucin and plasminogen The Centre of Applied Science for Health is funded by
[57-59] and as a result may play a direct role in the microbial the Programme for Research in Third Level Institutions
host response for certain pathogens. This may be an impor- (PRTLI) Cycle 4, supported by the European Union Re-
tant criterion in discerning the most protective antigens
gional Development Plan, the Irish Government National
among all those identified in an immunoproteomics study.
Development Plan 2007-2013 and administered by the
Immunoreactogenicity needs to be considered alongside the
Higher Education Authority in Ireland. RD is funded by Sci-
potential pathogenicity of any individual protein, as protec-
ence Foundation Ireland. SMcC is a member of the EU
tive vaccine antigens tend to be those that play a role in
COST Action BM1003: Microbial cell surface determinants
pathogenesis. Many immunoreactive proteins identified in
of virulence as targets for new therapeutics in cystic fibrosis.
814 Current Protein and Peptide Science, 2012, Vol. 13, No. 8 Dennehy and McClean
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Received: June 28, 2012 Revised: July 28, 2012 Accepted: August 03, 2012